Osteoporosis is a disease characterized by low peak bone mass leading to higher fragility where fractures occur after minimal trauma. Therefore, of considerable importance in the prevention and treatment of osteoporosis are studies that focus on understanding the mechanisms regulating bone accretion during critical windows in time when rapid bone accretion takes place. To this end, our studies involving genetic mouse models that are deficient in thyroid hormone (TH) and/or growth hormone (GH) action have provided unequivocal evidence that there is an important window of time, prior to puberty, when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Therefore, studies on the mechanism by which TH regulates skeletal growth during the unique window in time during the prepubertal growth period may have future ramifications concerning strategies to increase peak bone mass in children with growth disorders. Our studies during the past grant period have provided for the first time irrevocable evidence that the rapid increase in TH levels that occur during the second week of postnatal life is obligatory for the initiation and progression of secondary ossification center (SOC) in mice. Our studies also have led to an unexpected discovery that TH treatment causes several-fold increase in osterix (Osx) expression in chondrocytes in vitro and that Osx mRNA levels were severely compromised in bones of TH deficient mice compared to control mice. Based on these and other data, we propose a novel mechanism that several fold elevated levels of TH during second week of prepuberal growth period promotes conversion of chondrocytes in the SOC into bone matrix producing chondroblast/osteoblast cells via Osx-dependent mehcanism and thereby initiate bone formation. To determine the mechanism and role of TH-induced Osx in regulating endochondral ossification at the SOC, we propose to test the following 3 hypotheses in this continuation grant application. To test hypothesis 1 that TH regulates Osx expression in vivo during prepubertal growth period, we will evaluate the effect of TH treatment on expression of Osx in SOC using a TH deficient mouse model that expresses GFP:Cre under the control of Osx promoter. To test hypothesis 2 that TH-induced increase in Osx expression is a key determinant in SOC, we will evaluate the skeletal phenotype of tamoxifen-inducible cartilage-specific Osx knockout mice that have been made hypothyroid and treated with TH or vehicle during prepubertal growth period. To test hypothesis 3 that TH effect on Osx gene expression is mediated via binding of TR?1 to novel transcriptional element/s within the Osx promoter, we will identify the TH regultaory region and perform ChIP and EMSA. Because of the established importance of TH in regulating skeletal growth in both humans and experimental animals, the confirmation of our hypothesis that TH is indispensable for bone formation at SOC and that TH effects on bone are mediated via upregulation of Osx expression in chondrocytes, could lead to the development of effective therapies, based on TH action, to treat children with growth disorders during a window in time when rapid growth occurs. PUBLIC HEALTH RELEVANCE: This study will test a novel hypothesis that thyroid hormone-induced increase in osterix expression in epiphyseal chondrocytes leads to transdifferentiation of chondrocytes into bone matrix producing chondro/osteoblasts and, thereby, initiates endochondral ossification at the secondary ossification centers during the prepubertal growth period when thyroid hormone levels are elevated. Human clinical studies and transgenic mouse studies have established that thyroid hormone is an extremely important molecule in the regulation of the bone formation process. Therefore, an understanding of the mechanism by which epiphyseal chondrocytes are converted into chondro/osteoblasts could lead to the development of novel therapeutic strategies to heal non-union fractures by promoting conversion of chondrocytes at the fracture site into chondro/osteoblasts so that fracture healing can occur at a faster pace as in the case of secondary ossification centers.